Hepatitis E virus, known previously to cause much acute viral hepatitis in developing countries, has recently been identified as a cause of sporadic hepatitis in industrialized countries. It also has been found to cause chronic hepatitis in severely immune-compromised persons such as those receiving organ transplants. Inability to propagate the virus efficiently in cell culture or in small animal models has inhibited attempts to characterize its molecular biology or identify its pathogenic determinants. Two genotypes of the virus infect humans only, but the remaining two genotypes that infect humans are zoonotic and swine are a known reservoir. It seems certain that other animal reservoirs for human HEV must exist but they have yet to be identified and the biological factors that permit some strains to cross species boundaries are completely unknown. In FY2010, we isolated a new strain of genotype 3 HEV from the stool of a chronically infected patient and serially passaged this new virus strain in cultured human hepatoma cells and adapted it to grow relatively efficiently in these cells. During the adaptation process we discovered that a recombinant HEV with a 174 nucleotide insertion as well as numerous point mutations was selected.We made an infectious cDNA clone of this virus. In FY2012 we mutated the infectious cDNA encoding our cell culture-adapted virus ( P6, Kernow strain, genotype 3) in order to study virus host range and protein function. Surprisingly, we found that the viral ORF3 protein, which was required for efficient virus egress in 4 other cell lines, was not needed for cell-to cell spread of the P6 virus in HepG2/C3A cells suggesting that the virus may utilize an alternative exit pathway in these cells. We also showed that the cell culture-adaptive point mutations in P6 virus lost their effect if a previously identified 174 nucleotide insertion was removed. In FY2012, we identified a number of non-hepatic cell lines that supported replication of P6 genomes and characterized them for their ability to be infected, produce infectious virus, and permit virus egress. This was done to identify possible substrates for producing a vaccine strain of HEV. Cross-genotype chimeric viruses were constructed which were tested for their host-range preference on human and swine cells in order to determine the basis for the zoonotic aspect of genotype 3 infections. In FY2012, we examined the cerebral spinal fluid (CSF) and urine from a second chronically-infected patient and were able to demonstrate that HEV was present in both fluids. This is this first report of HEV in human urine. We sequenced these RNAs and showed that the virus in the CSF contained an inserted sequence, analogous to that in P6, which was present in a subpopulation of genomes in the feces and serum, whereas the urine lacked the insert. We also obtained evidence that this insert, like that in P6 virus, promoted the growth of the virus in cell culture. Additionally, we were able to show that P6 virus, but not genotype 1 virus, could infect and replicate in cultured neural cells, a result which is compatible with the recovery of virus from the CSF and which supports the suggestion that there are extra-hepatic sites of HEV replication. Ribavirin has been used to treat a small number of patients chronically-infected with genotype 3 HEV. In FY2012, we therefore tested genotypes 1, 2, and 3 for sensitivity to ribavirin in cell-culture and were able to show all 3 genotypes were sensitive,meaning ribavirin may be an appropriate drug of choice for treatment of HEV infection by any genotype. We continued our collaboration with Andrew Firth and further characterized the new cis-reactive element we discovered in the P6 genome.